U.S. patent application number 10/855745 was filed with the patent office on 2004-12-30 for soluble clca-1 and antagonists to clca-1.
Invention is credited to Bowman, Michael, Goldman, Samuel J., Long, Andrew J., Sypek, Joseph P., Wood, Clive R..
Application Number | 20040265314 10/855745 |
Document ID | / |
Family ID | 33490702 |
Filed Date | 2004-12-30 |
United States Patent
Application |
20040265314 |
Kind Code |
A1 |
Long, Andrew J. ; et
al. |
December 30, 2004 |
Soluble CLCA-1 and antagonists to CLCA-1
Abstract
The invention relates to methods of treating a disease or
condition, wherein expression or activity of soluble CLCA1 is
up-regulated, by administering inhibitors of soluble CLCA1. The
invention also relates to methods of isolating soluble CLCA1 from a
bodily fluid.
Inventors: |
Long, Andrew J.;
(Chelmsford, MA) ; Wood, Clive R.; (Boston,
MA) ; Bowman, Michael; (Westwood, MA) ;
Goldman, Samuel J.; (Acton, MA) ; Sypek, Joseph
P.; (Woburn, MA) |
Correspondence
Address: |
FINNEGAN, HENDERSON, FARABOW, GARRETT & DUNNER
LLP
1300 I STREET, NW
WASHINGTON
DC
20005
US
|
Family ID: |
33490702 |
Appl. No.: |
10/855745 |
Filed: |
May 27, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60474174 |
May 28, 2003 |
|
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|
Current U.S.
Class: |
424/146.1 ;
514/1.7; 514/1.8; 514/19.1 |
Current CPC
Class: |
A61P 11/06 20180101;
A61K 2039/505 20130101; C07K 14/47 20130101; A61K 38/00 20130101;
G01N 2500/04 20130101; G01N 33/6872 20130101; C07K 16/28 20130101;
A61P 11/00 20180101; A61P 43/00 20180101 |
Class at
Publication: |
424/146.1 ;
514/002 |
International
Class: |
A61K 039/395; A61K
038/00 |
Claims
What is claimed is:
1. A method of treating a subject having a disease or condition
wherein CLCA1 expression or activity is elevated in the subject
compared to an individual without the disease or condition, said
method comprising administering to said subject a therapeutically
effective amount of at least one agent which inhibits or prevents
soluble CLCA1 association with a cell membrane molecule, thereby
treating the disease or condition.
2. The method of claim 1, further comprising administering a
therapeutically effective amount of a second agent, wherein in said
second agent is an agent known to effectively treat the disease or
condition and wherein said second agent does not inhibit or prevent
soluble CLCA1 association with a cell membrane.
3. The method of claim 1 or 2, wherein the disease or condition is
asthma.
4. The method of claim 1 or 2, wherein the disease or condition is
COPD.
5. The method of claim 1, wherein at least one agent is a
protein.
6. The method of claim 1, wherein at least one agent is an
antibody.
7. The method of claim 1, wherein at least one agent is a small
molecule.
8. A method of isolating CLCA1 comprising obtaining a fluid from a
subject; contacting said fluid with a specific binding partner of
CLCA1 such that a complex between CLCA1 and the specific binding
partner forms; isolating the complex of CLCA1 and the specific
binding partner of CLCA1 from said fluid; and disrupting the
complex of CLCA1 and the specific binding partner of CLCA1 such
that CLCA1 is isolated.
9. The method of claim 8 wherein the fluid is a bodily fluid.
10. The method of claim 9, wherein the bodily fluid is sputum.
11. The method of claim 9, wherein the bodily fluid is obtained
from a mucosal surface.
12. The method of claim 8, wherein the fluid is bronchial alveolar
lavage.
13. The method of claim 8, wherein the specific binding partner is
a protein.
14. The method of claim 8, wherein the specific binding partner is
an antibody.
15. The method of claim 8, wherein the specific binding partner is
linked to a solid support.
Description
FIELD OF THE INVENTION
[0001] The invention relates to soluble Calcium Activated Chloride
Channel 1 (CLCA1) and antagonists which inhibit the activity of
soluble CLCA1. The invention relates in part to methods of
isolating and using soluble CLCA1 and antagonists to soluble CLCA1
to treat diseases and conditions associated with the abnormal
activity of CLCA1.
BACKGROUND OF THE INVENTION
[0002] Ion transport across the plasma membrane is critical for
maintaining the normal physiology of the cell. Ion transport across
the plasma membrane is mediated by a variety of membrane bound
proteins which act as channels and pumps. Dysfunctional ion
channels or pumps will lead to a disease state. Cystic fibrosis is
a disease resulting from a defect in a cAMP-mediated chloride
channel, CFTR (Welsh et al. 1993, Cell 73:1251). The physiological
manifestation of cystic fibrosis includes airway obstruction
resulting from thick secretions of mucus into the airways of the
lung and gastrointestinal tract and the subsequent colonization of
the lung airways by pathogenic microorganisms (Clarke et al. 1992,
Science 257:1125; Clarke et al. 1994, Proc Natl Acad Sci USA
91:479; Eng et al. 1996, Ped Pulmonol 21:77) and mucus plugging of
pancreatic ducts of the gastrointestinal tract (WO 01/54685). The
association of cystic fibrosis with aberrant ion transport has lead
investigators to hypothesize that dysfunctional ion transport might
be related to other diseases with similar symptoms. Thus,
dysfunctional ion transport has been implicated in diseases such as
asthma and chronic obstructive pathway disease (COPD), i.e.,
emphysema and chronic bronchitis.
[0003] CLCA1, and its murine homolog, gob-5, are putative calcium
activated chloride channels (WO 99/44620). Both have been
implicated in the pathology associated with asthma. Asthma is
characterized by a hypersensitivity to environmental allergens
which is associated with an inflammatory response and the increased
production of mucin (WO 01/54685; WO 99/44620). Expression of CLCA1
and gob-5 are up-regulated in response to allergen challenge.
Expression has also been linked to mucin overproduction (Hoshino et
al. 2002, Am J Respir Crit Care Med 165:1132). Over expression of
CLCA1 and gob-5 has been shown to induce expression of MUC5AC, a
mucin gene, in a muco-epidermal cell line. Additionally, adenovirus
mediated antisense therapy has abrogated the effects of gob-5
hyper-responsiveness and mucin production in an in vivo mouse model
(Nakanishi et al. 2001, Proc Natl Acad Sci USA 98:5175).
[0004] It has been suggested that CLCA1 is expressed as a 125 kD
precursor which is cleaved to form a 90 kD subunit and a 37 kD
subunit which associate on the cell surface to form an active ion
channel. It has also been suggested that the 90 kD subunit forms
four transmembrane domains. It has further been suggested that
expression of CLCA1 is associated with chloride ion flux across the
plasma membrane and that non-selective chloride channel inhibitors
such as niflumic acid,
4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, and
dithiothreitol will abrogate this effect. (Gruber et al. 1999,
Genomics 54:200).
SUMMARY OF THE INVENTION
[0005] The present invention is based at least in part on the
discovery that CLCA1 is a secreted protein that can interact, e.g.,
associate with an extra-cellular molecule, e.g., a cell membrane or
a cell membrane associated molecule. Because CLCA1 is a secreted
protein it provides a convenient target for treating diseases
associated with the increased expression or activity of CLCA1,
e.g., atopic diseases such as asthma or COPD-like diseases which
are characterized by high levels of mucin.
[0006] The invention relates to a method of treating a subject
having a disease or condition wherein CLCA1 expression or activity
is elevated in the subject compared to an individual without the
disease or condition, said method comprising administering to said
subject a therapeutically effective amount of at least one agent
which inhibits or prevents an interaction or association of soluble
CLCA1 with an extra-cellular molecule, e.g., association with a
cell membrane molecule, thereby treating the disease or condition.
Therefore, the invention provides for new methods of treating
atopic diseases or conditions such as asthma or diseases such as
COPD.
[0007] The invention relates to a method of modulating CLCA1
expression or activity in a cell, e.g. reducing CLCA1 expression or
activity, said method comprising administering an effective amount
of at least one agent which inhibits or prevents an interaction or
association of soluble CLCA1 with an extra-cellular molecule, e.g.,
association with a cell membrane molecule, thereby reducing the
expression or activity of CLCA1.
[0008] The agent can be an antibody or antigen binding fragment
thereof which binds to CLCA1; a small molecule; a peptide or a
peptide mimetic.
[0009] The invention further relates to a method of isolating CLCA1
comprising obtaining a fluid from a subject; contacting said fluid
with a specific binding partner of CLCA1 such that a complex
between CLCA1 and the specific binding partner forms; isolating the
complex of CLCA1 and the specific binding partner of CLCA1; and
disrupting the complex of CLCA1 and the specific binding partner of
CLCA1 such that CLCA1 is isolated.
[0010] Additional objects and advantages of the invention will be
set forth in part in the description which follows, and in part
will be obvious from the description, or may be learned by practice
of the invention. The objects and advantages of the invention will
be realized and attained by means of the elements and combinations
particularly pointed out in the appended claims.
[0011] It is to be understood that both the foregoing general
description and the following detailed description are exemplary
and explanatory only and are not restrictive of the invention, as
claimed.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 depicts the primary amino acid sequence of human
CLCA1. The VWA domain is underlined and the putative N-linked
glycosylation sites are marked with an asterisk. The signal
sequence is indicated by SS (SEQ ID NO: 10).
[0013] FIG. 2 demonstrates that both CLCA1 and gob-5 proteins are
secreted from COS cells transfected to express the proteins.
[0014] FIG. 3 is a Western blot demonstrating the presence of CLCA1
in conditioned media, cell lysate and membrane preparation of cells
transfected to express CLCA1.
[0015] FIG. 4 depicts a hydrophobicity analysis of CLCA1 compared
to other known membrane bound and secreted proteins using the
computer program TMHMM. CLCA1 does not contain any
membrane-spanning alpha helices.
[0016] FIG. 5 depicts a hydrophobicity analysis of CLCA1 compared
to other known membrane bound and secreted proteins using the
computer program Split 4.0. CLCA1 does not contain any
membrane-spanning alpha helices.
[0017] FIG. 6 is a Western blot demonstrating that CLCA1 is
secreted as a multimer.
[0018] FIG. 7 demonstrates that CLCA1 is highly glycosylated.
PNGase treatment reduces the size of the secreted protein.
[0019] FIG. 8 is a Western blot demonstrating that the subunits of
CLCA1 are not linked by a disulfide bond.
[0020] FIG. 9 demonstrates that CLCA1 is present in the BAL fluid
of asthmatic and non-asthmatic patients.
[0021] FIG. 10 demonstrates that CLCA1 activates the MUC5AC
promoter in a dose dependent manner.
[0022] FIG. 11 depicts a schematic diagram of CLCA1 deletion
mutants showing the location of VWA domain, MIDAS domain residues
and proposed cleavage sites.
[0023] FIG. 12a shows MUC5AC-luciferase reporter induction by
over-expression of CLCA1 or gob-5 in A549 cells. CLCA and gob-5
show a 2 fold induction of MUC5AC-luciferase reporter activity as
compared to vector transfected controls. FIG. 12b shows a similar
result in HEK293 cells.
[0024] FIG. 13 shows whole-cell patch clamp recording of
transiently expressed CLCA1 and gob-5 in HEK 293 and A549 cells.
FIG. 13(a) shows current-voltage relationship of transiently
expressed CLCA1 in HEK 293 cells (left panel) and in A549 cells
(right panel) at 0.5 M free Ca2+ in the pipette solution (n=6-8 in
each group). FIG. 13(b) shows calcium-dependent fold increase in
whole-cell current density in HEK 293 cells, transiently expressing
hCLCA1 (left panel) and gob-5 (right panel) (n=3-8 in each group).
Similar data was also observed in A549 (data not shown).
[0025] FIG. 14 is a western blot showing CLCA1 and gob-5 expression
in HEK293 cells and in A549 cells.
DESCRIPTION OF THE EMBODIMENTS
[0026] Antibody, as used herein, means an immunoglobulin or a part
thereof, and encompasses any polypeptide comprising an
antigen-binding site regardless of the source, method of
production, or other characteristics. The term includes for
example, polyclonal, monoclonal, monospecific, polyspecific,
humanized, single-chain, chimeric, synthetic, recombinant, hybrid,
mutated, and CDR-grafted antibodies. A part of an antibody can
include any fragment which can bind antigen, for example, an Fab,
F(ab').sub.2, Fv, scFv.
[0027] The term "CLCA1," as used herein, refers to a protein that
is not a membrane spanning protein. It can be covalently or
non-covalently associated with a membrane or membrane associated
molecule or a secreted molecule.
[0028] CLCA1 activity, as used herein, refers to modulation of an
ion channel, e.g., a chloride channel.
[0029] The term "mucin", as used herein, refers to a fluid, e.g.,
secretion containing carbohydrate rich glycoproteins. Mucin can be
secreted from the goblet cells of the intestines, the submaxillary
glands or other glandular cells.
[0030] "Small molecule," as used herein, refers to any molecule
having a molecular weight of less than 25 kD.
[0031] "Specific binding partner," as used herein, means a first
molecule that specifically interacts with a second molecule. The
interaction can be the result of a covalent bond, or a non-covalent
bond, e.g., an ionic bond, a charge-charge interaction, a
hydrophobic interaction, a hydrogen bond, or van der Waal's
forces.
[0032] The term "subject," as used herein, means a human or a
non-human mammal, e.g., a dog, a cat, a mouse, a rat, a cow, a
sheep, a pig, a goat, a non-human primate.
[0033] The terms "treat," "treatment," and "treating," as used
herein refers to any of the following: the reduction in severity of
a disease or condition; the reduction in the duration of a disease
course; the amelioration of one or more symptoms associated with a
disease or condition; the provision of beneficial effects to a
subject with a disease or condition, without necessarily curing the
disease or condition; the prophylaxis of one or more symptoms
associated with a disease or condition.
[0034] "Up-regulated," as used herein, refers to a protein
expressed at levels in a subject having a disease or condition that
are greater than the levels of the same protein in an individual
who does not have the disease or condition.
[0035] Methods of Treatment
[0036] The invention relates to a method of treating a disease or
condition where expression or activity of CLCA1 is up-regulated,
comprising administration of at least one agent which inhibits or
prevents CLCA1 association with a cell membrane molecule.
[0037] CLCA1 is a cellular protein implicated in diseases and
conditions involving the up-regulation of mucin, e.g., atopic
conditions such as asthma (FIG. 1; WO 99/44620). The present
invention is based at least in part on the discovery that CLCA1 is
a secreted protein that can interact, e.g., associate with an
extra-cellular molecule, e.g., a cell membrane or a cell membrane
associated molecule. By providing agents which inhibit or prevent
CLCA1 from associating with membrane molecules and thus potentially
increasing mucin production, the invention provides for a method of
treating a disease or condition characterized by the up-regulation
of mucin.
[0038] Thus, the invention relates to a method of treating a
disease or condition wherein CLCA1 expression or activity is
enhanced, e.g., up-regulated. The disease or condition can be an
atopic or allergic disorder, e.g., asthma. The disease or condition
can be any disease or condition wherein mucin levels are enhanced,
e.g., mucin production and/or secretion is up-regulated. The
disease or condition can be cystic fibrosis or COPD, i.e.,
emphysema, chronic bronchitis. The disease or condition can be one
effecting any mucosal surface, e.g., a pulmonary surface or a
gastro-intestinal surface. The disease or condition can be an
infectious disease, e.g., a viral infection or a bacterial
infection. The disease or condition can be either chronic or
acute.
[0039] The methods of the invention employ an agent that
specifically inhibits the activity of CLCA1, e.g., soluble CLCA1.
In one embodiment, the agent binds directly to CLCA1, e.g. soluble
CLCA1. In another embodiment, the agent inhibits, reduces or
prevents CLCA1, e.g., soluble CLCA1 binding to an extra-cellular
molecule, e.g., a membrane associated protein or lipid.
[0040] An agent which inhibits CLCA1 activity can include, but is
not limited to, a protein or a protein fragment, e.g., a peptide.
The protein or protein fragment can be recombinantly produced using
known techniques, see, e.g., Sambrook et al. 1989, Molecular
Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory
Press. Alternatively, the protein or protein fragment can be made
by chemical synthesis using known techniques, see, e.g., Merrifield
1973, Chemical Polypeptides, (Katsoyannis and Panayotis eds.) pp.
335-61; Merrifield 1963, J. Am. Chem. Soc. 85:2149; Davis et al.
1985, Biochem. Intl. 10:394; Finn et al. 1976, The Proteins
(3.sup.rd ed.) 2:105; Erikson et al. 1976, The Proteins (2nd ed.)
2:257; U.S. Pat. No. 3,941,763. The protein or protein fragment can
be derivatized, e.g. pegylated. The protein or protein fragment can
be fused to another protein to make a fusion protein, e.g., a GST
fusion protein, an Fc fusion protein. The protein or protein
fragment can be a glycoprotein or a lipoprotein. In one embodiment,
the agent which inhibits CLCA1 activity is a CLCA1 specific binding
partner.
[0041] The agent which inhibits CLCA1 activity can be a small
molecule. The small molecule can be an organic molecule or an
inorganic molecule. The small molecule can be synthesized using
known techniques see, e.g., Morrison and Boyd, 1987, Organic
Chemistry Fifth Edition, Allyn and Bacon, Newton, Mass.
[0042] In one specific embodiment the agent is an antibody or a
fragment of an antibody. A fragment of an antibody can include, but
is not limited to Fab, F(ab').sub.2, or a single chain Fv. The
antibody can be a monoclonal antibody or a polyclonal antibody. In
general, antibodies can be made, for example using traditional
hybridoma techniques (Kohler and Milstein 1975, Nature 256:495),
recombinant DNA methods (U.S. Pat. No.4,816,567), or phage display
performed on antibody libraries (Clackson et al. 1991, Nature
352:624; Marks et al. 1991 J Mol Biol, 222:581). For other antibody
production techniques, see, Antibodies: A Laboratory Manual, eds.
Harlow et al., 1988, Cold Spring Harbor Laboratory.
[0043] In vitro systems may be designed to identify compounds
capable of inhibiting CLCA1 activity, e.g., by binding to CLCA1.
The assay can involve preparing a reaction mixture of CLCA1 and a
test compound under conditions, and for a time, sufficient to allow
a complex to form. The complex can be a transient complex and the
complex can be removed or detected in the reaction mixture. CLCA1
can be anchored to a solid phase, e.g., a bead, a microtiter plate,
and then reacted with the test agent and the complex detected.
Alternatively, the test agent can be anchored to a solid phase, and
reacted with CLCA1 and the complex detected. Unreacted reagents can
be washed away. The non-anchored species can be labeled directly or
indirectly. Labels can include, for example, but not as a
limitation, dyes, chromophores or radioactivity. The label can be
conjugated to an antibody specific to the test agent or CLCA1.
[0044] In one embodiment, mucin production can be used to measure
CLCA1 activity in the presence or absence of a test CLCA1
inhibitory agent. Mucin production can be measured in vitro using a
muco-epidermal cell line, e.g., NCl--H292 transfected with the
human CLCA1 gene (see Nakanishi et al. 2001, Proc Natl Acad Sci USA
98(9):5175). A test agent inhibitor can be added to the media and
mucin production monitored. Mucin production can be monitored using
techniques well known in the art, e.g., ELISA, SDS-PAGE, or Western
blot.
[0045] The CLCA1 inhibitor can be administered intravenously,
subcutaneously, sublingually, intramuscularly, orally, buccally,
nasally, rectally, or via pulmonary route. The CLCA1 inhibitor can
be implanted within or linked to a biopolymer solid support that
allows for the slow release of the chimeric protein.
[0046] The CLCA1 inhibitor can be administered in combination with
a therapeutically effective amount of a second agent known in the
art for the treatment of a condition or disease. As an example, but
not as a limitation, asthma can be treated with a CLCA1 inhibitor
and a beta-receptor agonist or a cortico-steroid.
[0047] The dose of the CLCA1 inhibitor will vary depending on the
subject and upon the particular route of administration used.
Dosages can range from 0.1 to 100,000 .mu.g/kg body weight. In one
embodiment, the dosing range is 1-10 mg/kg. The CLCA1 inhibitor can
be administered continuously or at specific timed intervals. In
vitro assays may be employed to determine optimal dose ranges
and/or schedules for administration. Additionally, effective doses
may be extrapolated from dose-response curves obtained from animal
models, e.g., murine asthma (see Nakanishi et al. 2001, Proc Natl
Acad Sci USA 98(9):5175).
[0048] The invention also relates to a pharmaceutical composition
comprising CLCA1 inhibitors and a pharmaceutically acceptable
carrier or excipient. Examples of suitable pharmaceutical carriers
are described in Remington's Pharmaceutical Sciences by E. W.
Martin. Examples of excipients can include starch, glucose,
lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel,
sodium stearate, glycerol monostearate, talc, sodium chloride,
dried skim milk, glycerol, propylene, glycol, water, ethanol, and
the like. The composition can also contain pH buffering reagents,
and wetting or emulsifying agents.
[0049] For oral administration, the pharmaceutical composition can
take the form of tablets or capsules prepared by conventional
means. The composition can also be prepared as a liquid for example
a syrup or a suspension. The liquid can include suspending agents
(e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible
fats), emulsifying agents (lecithin or acacia), non-aqueous
vehicles (e.g., almond oil, oily esters, ethyl alcohol, or
fractionated vegetable oils), and preservatives (e.g., methyl or
propyl-p-hydroxybenzoates or sorbic acid). The preparations can
also include flavoring, coloring and sweetening agents.
Alternatively, the composition can be presented as a dry product
for constitution with water or another suitable vehicle.
[0050] For buccal and sublingual administration the composition may
take the form of tablets or lozenges according to conventional
protocols.
[0051] For administration by inhalation, the compounds for use
according to the present invention are conveniently delivered in
aerosol form, e.g., an aerosol spray from a pressurized pack or
nebulizer, with a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoromethane, carbon dioxide or other suitable gas.
In the case of a pressurized aerosol the dosage unit can be
determined by providing a valve to deliver a metered amount.
Capsules and cartridges of, e.g., gelatin for use in an inhaler or
insufflator can be formulated containing a powder mix of the
compound and a suitable powder base such as lactose or starch.
[0052] The pharmaceutical composition can be formulated for
parenteral administration (i.e., intravenous or intramuscular) by
bolus injection. Formulations for injection can be presented in
unit dosage form, e.g., in ampoules or in multidose containers with
an added preservative. The compositions can take such forms as
suspensions, solutions, or emulsions in oily or aqueous vehicles,
and contain formulatory agents such as suspending, stabilizing
and/or dispersing agents. Alternatively, the active ingredient can
be in powder form for constitution with a suitable vehicle, e.g.,
pyrogen free water.
[0053] The pharmaceutical composition can also be formulated for
rectal administration as a suppository or retention enema, e.g.,
containing conventional suppository bases such as cocoa butter or
other glycerides.
[0054] Methods of Isolating CLCA1
[0055] CLCA1 is a secreted soluble protein. Accordingly, it is
readily isolated from any fluid containing it by contacting the
fluid with a specific binding partner of CLCA1.
[0056] CLCA1 can be isolated from any biological fluid. A
biological fluid can include any body fluid or secretion. In one
embodiment, CLCA1 is isolated from sputum. In another embodiment,
CLCA1 is isolated from bronchial alveolar lavage. The bodily fluid
can be obtained from any mucosal surface. In one embodiment, the
mucosal surface is a pulmonary surface or a rectal surface.
[0057] A specific binding partner can include, but is not limited
to, a protein or a protein fragment, e.g., a peptide. The protein
or protein fragment can be derivatized, e.g., pegylated. The
protein or protein fragment can be part of a fusion protein, e.g.,
a GST fusion protein, an Fc fusion protein. The protein or protein
fragment can be a glycoprotein or a lipoprotein. The specific
binding partner can be a small molecule. The small molecule can be
an organic molecule or an inorganic molecule. In one specific
embodiment the agent is an antibody or a fragment of an antibody. A
fragment of an antibody can include, but is not limited to Fab,
F(ab').sub.2, or a single chain Fv. The antibody can be a
monoclonal antibody or a polyclonal antibody.
[0058] Soluble CLCA1 can be isolated by reacting it with a specific
binding partner under a first set of conditions such that a complex
forms between CLCA1 and the specific binding partner. The reaction
can occur in a liquid phase and the complex isolated by contacting
it with an antibody which recognizes the specific binding partner.
Alternatively, the specific binding partner can be linked to a
solid support, either directly or indirectly, e.g., by an antibody
which recognizes the specific binding partner. The solid support
can be, for example, but is not limited to, a bead, a microtiter
plate, a matrix of a column, a membrane, or a monolith. Once the
complex has formed, the reaction conditions can be changed, e.g.,
by changing the pH, or by changing the salt concentration of the
solution containing the complex, to a second set of conditions such
that the complex is disrupted. The specific binding partner remains
bound to the solid support, thus providing a solution containing
isolated CLCA1.
EXAMPLES
Example 1
Transient Expression of CLCA1 and Gob-5 in COS-7 and HEK293
Cells
[0059] COS and HEK293 cells were maintained in Dulbecco's modified
Eagle's (DME) medium supplemented with 10% (v/v) fetal calf serum
(FCS), 50 units/ml penicillin, 50 .mu.g/ml streptomycin and 2 mM
glutamine and grown in a humidified atmosphere at 37.degree. C. At
16 hours prior to transfection, COS cells were seeded in a 100mm
tissue culture treated dish at 4.times.10.sup.6 cells per dish.
After cells had reached approximately 70% confluence, 10 .mu.g of
DNA was mixed with Lipofectamine 2000 reagent (Invitrogen,
Carlsbad, Calif.) in Opti-MEM serum free medium (Life Technologies,
Carlsbad, Calif.) as described in the manufacturer's instructions.
Lipofectamine 2000/DNA mixture was added to the culture medium of
the COS cells for 6 hours at 37.degree. C. After the incubation
period, the medium in the plate was replaced with complete medium
(DME 10% FCS). Cells were allowed to incubate at 37.degree. C. for
24-48 hours after which the conditioned media and cell lysates were
harvested for biochemical analysis.
Example 2
Radioactive Incorporation into Heterologously Expressed Protein
[0060] COS cell monolayers transfected with CLCA1, gob-5, or
pcDNA3.1 vector control were incubated for 20 minutes in serum free
DME without cysteine or methionine. The cells were then labeled
with 300 .mu.Ci of Promix (Amersham, Piscataway, N.J.), a mixture
of .sup.35S labeled cysteine and methionine, per 100 mm dish for 30
minutes. The radioactive amino acid containing medium was removed
and the cells were washed 3 times with PBS. Radioactive waste was
disposed of in accordance Nuclear Regulatory Commission guidelines.
DME with 10% v/v fetal calf serum was added and cells were
incubated for 2 to 6 hours at 37.degree. C. At these time points,
conditioned media and cell lysates were harvested. Incorporated
radioactivity was determined by TCA precipitation as follows. For
each sample,1 .mu.l was blotted onto a small piece of Whatman 3 MM
paper. Sample paper was immersed in ice-cold 10% w/v trichloracetic
acid (TCA) for 10 minutes. The samples were washed 3 successive
times for 5 minutes each with ice-cold ethanol and then allowed to
air dry. Once dry, paper samples were added to 10 ml of Scinti-Safe
(Fisher Scientific, Hanover Park, Ill.) scintillation fluid in
scintillation vials. Levels of radioactivity were measured in a
Beckman Scintillation Counter.
Example 3
Harvesting of Cell Lysates
[0061] Cell monolayers were washed twice with ice-cold, calcium and
magnesium free, phosphate buffered saline (PBS). Cells were removed
from the dish using 0.25% (v/v) Trypsin-EDTA (Gibco, Carlsbad,
Calif.) and counted using a hemacytometer. The cells were washed
twice with ice cold PBS and lysed in NP40 lysis buffer (1% NP-40,
50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 10% glycerol) for 15 minutes
on ice at a concentration of 0.5.times.10.sup.6 cells/10 .mu.l of
lysis buffer. The resulting lysate was cleared by
microcentrifugation at 15000 RPM for 15 minutes and then stored at
-80.degree. C. until used in Western blots.
Example 4
Membrane Preparations
[0062] COS cells grown in 100 mm tissue culture treated dishes were
transfected as described in Example 1. 48 hours after transfection,
cells were removed from the plate with trypsin and washed twice
with cold PBS in a 50 ml tube. The cells were resuspended in 4 ml
of 1 mM Tris pH 7.4 and left on ice for 20 minutes. The cell
suspension was homogenized with 10 strokes of a clean dounce
homogenizer. The samples were then spun at 2000 g in a Sorvall
Legend RT table top centrifuge for 10 minutes. The supernatant was
transferred to an ultracentrifuge tube and spun by
ultracentrifugation at 100,000 g for 1 hour. The resultant pellet
was suspended in 100 .mu.l of NP40 lysis buffer.
Example 5
Western Blotting
[0063] Samples were combined with 2.times. SDS sample buffer with
.beta.-mercapatoethanol and boiled for 2 minutes. After boiling,
the samples were loaded onto either a 4-20% gradient gel or 6% SDS
gel. Gels were then transferred to nitrocellulose using an
Invitrogen blotting apparatus (Invitrogen, Carlsbad, Calif.) and
transfer buffer as provided by Invitrogen (Invitrogen, Carlsbad,
Calif.). Gels were transferred for 2 hours at 30V under constant
current. Nitrocellulose blots were blocked in 5% non-fat dry milk
for 1 hour at room temperature. The blots were incubated with 0.5
.mu.g/mL peptide polyclonal antibody overnight at 4.degree. C.
After incubation with the primary antibody, the blots were washed 3
times for 10 minutes each in washing buffer (1% Triton.times.100
(v/v), 0.1% SDS (w/v) in PBS, calcium and magnesium free) and then
incubated for 30 minutes with mouse anti rabbit IgG conjugated to
horseradish peroxidase (Santa Cruz Biotech, Santa Cruz, Calif.).
The blots were washed 3 times for 10 minutes each in wash buffer
and developed with the chemiluminescence reagent, ECL. (Amersham,
Piscataway, N.J.). The blots were exposed to Kodak film and
developed using a film developer.
Example 6
Generation of Constructs
[0064] CLCA1 was isolated from human colon cDNA using polymerase
chain reaction (PCR). The 5' anchoring primer used in the reaction
was as follows: 5'-CGCAAGCTTG GGATGGGGCC ATTTMGAGT TCTGTGTTC-3'
(SEQ ID NO:1). The underlined sequence represents a HindIII
restriction endonuclease site. All constructs used this 5' primer.
The 3' primer used in deletion constructs were as follows: (1-708)
5'-CCCMGCTTT CACTACTTAT TMTTTCAGG TCTTGGTGG-3' (SEQ ID NO: 2),
(1-660) 5'-CCCAAOCTTT CACTACCTTG AGTAGACACC GTCATCCTTA G-3' (SEQ ID
NO: 3) and (1-462) 5'-CCCMGCTTT CACTATTGAT CTGMGCATA TGTCTGTAAA
CCTCCCTG-3' (SEQ ID NO: 4). PCR products were amplified with high
fidelity Taq DNA polymerase (Invitrogen, Carlsbad, Calif.).
Deletion constructs used wild-type CLCA1 as a template. The
template was denatured for 2 minutes at 96.degree. C. and then the
reaction proceeded as follows: the DNA was denatured at 94.degree.
C. for 30 seconds, annealed at 65.degree. C. for 1 minute, and
extended at 72.degree. C. for 1 minute. This cycle was repeated for
20 cycles. The resulting product was digested with HindIII.
pcDNA3.1 was digested with HindIII and dephosporylated with calf
intestinal alkaline phosphatase (New England Biolabs, Beverly,
Mass.). Fragments were ligated using Rapid DNA ligation kit (Roche,
Basel, Switzerland) and then transformed with One Shot chemically
competent E. coli (Invitrogen, Carlsbad, Calif.).
Example 7
Generation of Polyclonal Antibodies
[0065] Peptide polyclonal antibody generation was performed at
Invitrogen (Carlsbad, Calif.) according to the protocol described
herein. New Zealand white rabbits (age 3-9 months) were immunized
with KLH-peptide (see below) emulsified in Freund's Adjuvant by
subcutaneous injections at three dorsal sites (0.1 mg peptide per
immunization). Animals were bled by articular artery. Serum was
collected and titer was determined via free peptide ELISA. Five
hydrophilic peptides were selected based on extracellular domains
as previously described (Nakanishi et al. 2001, Proc Natl Acad Sci
USA 98:5175). The hyrdophillic peptides included 1CLC,
corresponding to amino acids 124-140, Gly Glu Lys Gly Glu Arg Ile
His Leu Thr Pro Asp Phe Ile Ala (SEQ ID NO: 5); 2CLC, corresponding
to amino acids 202-217, Glu Gin Asn His Asn Lys Glu Ala Pro Gin Lys
GIn Asn GIn Lys; 3CLC (SEQ ID NO: 6), corresponding to amino acids
478-495, Gly GIn Gly Val Ser Gin Arg Ser Ile Gin Leu Glu Ser Lys
Gly Leu Thr (SEQ ID NO: 7); 4CLC, corresponding to amino acids
702-719, lie Glu Asn Asp Glu lie GIn Trp Asn Pro Pro Arg Pro Glu
Ile (SEQ ID NO: 8); and 5CLC, corresponding to amino acids 819-833,
Lys Glu Ala Asn Ser Glu Glu Val Phe Leu Phe Lys Pro Glu Asn (SEQ ID
NO: 9).
Example 8
PNGase F Treatment
[0066] PNGase F was obtained from New England Biolabs (Beverly,
Mass.) and was used according to the manufacturer's instructions.
Samples were boiled in reducing buffer for 5 minutes.
Ethylphenyl-polyethylene glycol (NP-40) was added to a final
concentration of 1% (v/v) along with 1.times.G7 reaction buffer
(New England Biolabs, Beverly, Mass.)). 1 .mu.l of PNGase F was
added and incubated for 1 hour at 37.degree. C. 2.times. sample
buffer (Invitrogen, Carlsbad, Calif.) was then added and samples
were resolved on Tris-glycine gels as described in Example 5.
Example 9
MUC5AC Reporter Assay
[0067] A 1.6 kilobase fragment of the MUC5AC promoter was fused to
the firefly luciferase gene used as a reporter (MUC5AC-luc). A549
cells were transfected in 6-well tissue culture treated plates with
Fugene transfection reagent (Roche, Basel, Switzerland) using a
total of 2 .mu.g per well. 0.5 .mu.g of MUC5AC-luc construct was
used per well. Varying concentrations of CLCA1 or deletion
constructs were used to establish a dose response. Vector DNA
(pcDNA3.1) was used to complete DNA total to 2 .mu.g. The cells
were washed twice and transfected for 6 hours in serum free
Hams/F12 media (Life Technologies, Carlsbad, Calif.). After 6
hours, the media was changed to serum containing, i.e., 10% serum
Hams/F12. The cells were lysed in cell lysis solution provided with
the luciferase assay reagents (Promega, Madison, Wis.) and the
manufacturer's instructions were followed. The samples were
transferred in triplicate to a 96-well plate and were read on a
96-well luminometer (Turner Biosystems, Sunnyvale, Calif.).
Example 10
Computational Analysis of CLCA1
[0068] CLCA1 is a 914 amino acid polypeptide that contains nine
putative N-linked glycosylation sites (FIG. 1). A hydrophobic
stretch of 21 residues is predicted to be a secretory signal
sequence according to the algorithm SigCleave. The CLCA1
polypeptide sequence was also used to query the NCBI Conserved
Domain Database (http://www.ncbi.gov) and discovered to contain a
central von Willebrand factor A-domain (VWA) that spans 270 amino
acids. The VWA domain of CLCA1 contains an intact MIDAS motif, a
defined set of amino acids responsible for binding of divalent
metal cations.
[0069] CLCA1 was evaluated using the transmembrane hidden Markov
model (TMHMM) (Sonnhamer et al. 1998, Proc Sixth Int Conf on
Intelligent Systems for Molecular Biology, AAAI Press). The model
was developed as a tool to analyze membrane spanning alpha helices
and to predict not only membrane spanning segments but also
membrane topology as well. The TMHMM suggests that CLCA1 will have
no transmembrane segments just like the secreted protein, serum
albumin (FIG. 4). To ensure that the models are predictive, other
proteins with well understood transmembrane topology or solved
crystal structure were analyzed. The model consistently predicted
the 12 hydrophobic segments of CFTR and the defined hydrophobic
stretches of CLC from E. coli (Riordan et al. 1989, Science
254:1606; Dutzler et al. 2002, Nature 415:287). Based on another
hydrophobicity model, Split 4.0 (http://pref.effos.hr/split-4.0/),
CLCA1 is also predicted to be a soluble protein (FIG. 5).
Example 11
Heterologous Expression of CLCA1
[0070] To examine its cellular localization, CLCA1 was
heterologously expressed in COS-7 cells and HEK293 cells using the
mammalian expression vectors pcDNA3.1 and pDEST 12.2.
Overexpression of CLCA1 yielded a surprising 120 kD species in the
culture medium as measured by .sup.35S cys-met radioactive
incorporation (FIG. 2). The presence of these bands in the culture
media suggests CLCA1 is secreted. To confirm the identity of CLCA1,
peptide polyclonal antibodies were generated against peptides that
corresponded to extracellular domains as suggested by Gruber
(Gruber et al. 1999, Genomics 54:200) for use in western blot
analysis. The secreted phenotype was indeed confirmed by Western
blotting, which revealed a CLCA1 immunoreactive band of 120 kD in
conditioned media of both COS-7 transfected cells and HEK293
transfected cells (FIG. 3A). The presence of full length CLCA1 was
confirmed in the conditioned media. A 120 kD band and a 37 kD band
were also seen in the cell lysate by Western blot (FIG. 3B). These
bands were not present in vector control transfected cells.
[0071] Membrane preparations were analyzed to test whether the
bands present in the cell lysates are from the secretory pathway or
associated with the membrane. Both the 120 kD and the 37 kD bands
are associated with the membrane (FIG. 3C). Conditioned media
samples from vector and CLCA1 transfected HEK293 cells were
analyzed by mass spectrometry to verify the specificity of the
polyclonal antibodies. The CLCA1 immunoreactive bands in the
Western blots were confirmed to be full length CLCA1.
[0072] The conditioned media from both COS and HEK293 transfectants
were analyzed by Western blot under reducing and non-reducing
conditions to assess whether CLCA1 exists as a multimer in the
secreted milieu. Under reducing conditions, CLCA1 migrates at a
size of approximately 120 kD. Under non-reducing conditions, higher
species consistent with dimer formation become apparent (FIG. 6).
These observations were made using 4-20% gradient Tris-Glycine SDS
gels. To increase resolution, 6% Tris-Glycine gels were used. CLCA1
migrates, under non-reducing conditions, at sizes consistent with
dimer and multimer formation.
[0073] Based on the primary protein sequence, CLCA1 is predicted to
have a molecular weight of approximately 100 kD. Analysis by
SDS-PAGE reveals a species in the conditioned media that migrates
at 120 kD. CLCA1 has nine putative N-linked glycosylation sites
(FIG. 1). Secreted samples were treated with the N-glycosidase
PNGase F to determine the contribution of the N-linked sugars to
the molecular weight discrepancy. Treatment with PNGase F reduced
the size to the expected 100 kd (FIG. 7). This is consistent with
the fact that CLCA1 has nine putative N-linked glycosylation sites
(FIG. 1) and may be highly glycosylated.
[0074] Gruber et al. suggest that CLCA1 is cleaved from its full
length 120 kD product to yield a 90 kD and 37 kD product, which are
associated via disulfide linkage (Gruber et al. 1999, Genomics
54:200). To test whether the two subunits are linked via disulfide
linkage, lysates were run on Western blot under reducing and
non-reducing conditions (FIG. 8). Under non-reducing conditions,
the 37 kD species is still present. This observation would suggest
that disulfide linkages do not associate with the lower
subunit.
Example 12
Native Expression of Soluble CLCA1
[0075] Because the secreted phenotype of CLCA1 was seen primarily
in overexpression systems, there was a concern that the phenomenon
was an artifact of aberrant cellular processing. Gene expression
studies have suggested that CLCA1 is expressed in the lungs after
antigen challenge. As a result, bronchial alveolar lavage (BAL)
fluid from cynomologous monkeys challenged with Ascaris suum was
tested by Western blot for the presence of soluble CLCA1.
Immunoreactive bands of 90 kD and 120 kD were detected in the BAL
from both unchallenged and challenged states in the same
animals.
[0076] Next, samples of human BAL were tested from both asthmatic
and non-asthmatic patients. The results obtained from the human
samples were similar to the results obtained from the monkey BAL
samples. Consistently, there were two bands at 90 and 120 kD. There
was variability from patient to patient but overall, there did not
seem to be any difference in protein expression between asthmatic
and non-asthmatic samples (FIG. 9).
[0077] Based on expression studies and the physiologic observation
that induction of CLCA1 resulted in mucus production (Hoshino et
al. 2002, Am J Respir Crit Care Med 165:1132; Nakanishi et al.
2001, Proc Natl Acad Sci USA 98:5175), a reporter assay was
developed that used the MUC5AC gene promoter fused to the firefly
luciferase gene as an assay readout. A549 cells were used because
of their mucoepidermal and respiratory tract origins.
Overexpression of CLCA1 in A549 cells results in a dose dependent
increase in MUC5AC gene transcription as measured by luciferase
activity (FIG. 10).
Example 13
Generation of Constructs
[0078] Deletion and mutation constructs will be made to identify
the functional determinants of CLCA1 (FIG. 11). C-terminal deletion
constructs will have deleted amino acids 709-914 and 661-914, which
correspond to putative cleavage products as proposed by Gruber et
al. (Gruber et al. 1999, Genomics 54:200). Construct 1-462 will be
comprised of the N-terminus including the VWA domain. VWAdel will
be comprised of the entire molecule with the VWA domain deleted.
This will test the necessity of the VWA domain for the function of
CLCA1. Additionally, the MIDAS motif of the VWA domain, which is
necessary for divalent cation binding, will be mutated to eliminate
that binding.
Example 14
Expression of CLCA1 and Gob-5 in HEK293 and A549 Cells
[0079] The pneumocyte cell line A549 and the embryonic kidney cell
line HEK293 were each transfected with a plasmid, pcDNA3,
engineered to encode either CLCA1 or gob-5. Expression was
confirmed by Western blot. Samples were combined with 2.times. SDS
sample buffer with .beta.-mercapatoethanol and boiled for 2
minutes. After boiling, the samples were loaded on either 4-20%
gradient or 6% SDS gels. Gels were then transferred to
nitrocellulose using an Invitrogen blotting apparatus and transfer
buffer (Invitrogen, Carlsbad, Calif.). Gels were transferred for 2
hours at 30V under constant current. Nitrocellulose blots were
blocked in 5% (w/v) non-fat dry milk for 1 hour at room
temperature. Blots were incubated overnight at 4.degree. C. with
peptide polyclonal antibody which recognized either the N or C
terminus of CCLA1 or gob-5. After incubation with the primary
antibody, blots were washed 3 times for 10 minutes each in washing
buffer (1% (v/v) Triton X 100, 0.1% (w/v) SDS in PBS-calcium and
magnesium free) and then incubated for 30 minutes with mouse
anti-rabbit IgG conjugated to horseradish peroxidase. Blots were
washed 3 times for 10 minutes each in wash buffer and developed
with a chemiluminescence reagent (ECL reagent)(Amersham, Uppsala,
Sweden). Blots were exposed to Kodak film and developed using a
film developer. The results showed that both CLCA1 and gob-5 were
expressed (FIG. 14).
Example 15
CLCA1 and Gob-5 Induce MUC5AC Gene Transcription
[0080] A construct (MUC5AC-luc) comprising a 1.6 kb fragment of the
MUC5AC promoter fused upstream of the reporting gene, luciferase,
was used to determine if the overexpressed CLCA1 or gob-5 could
induce gene expression under the control of a mucin promoter.
[0081] A549 or HEK293 cells were transfected in 6-well tissue
culture treated plates with TransIT transfection reagent (Mirus
Corp., Madison, Wis.) using a total of 4 mg per well.
MUC5AC-luciferase construct (0.2 mg/well) was transfected into
HEK293 cells. 1 mg of MUC5AC-luciferase construct was used per well
for A549 cells. Twenty-four hours after transfection, the cells
were lysed in cell lysis solution provided with luciferase assay
reagents (Promega, Madison, Wis.) and the manufacturer's
instructions were followed. Samples were transferred in triplicate
to a 96-well plate and were read on a 96-well luminometer (Turner
Biosystems, Sunnyvale, Calif.). Luciferase data presented was
normalized for .beta.-galactosidase expression (HEK293 cells) or
per mg total protein (A549 cells).
[0082] The results demonstrated that CLCA1 and gob-5 both induced a
two fold increase in gene transcription compared to a negative
control (empty plasmid) (FIG. 12a A549 cells and 12b Hek293
cells).
Example 16
Measurement of Whole Cell Chloride Current Induction using
Physiological Ca Levels
[0083] A549 cells and HEK293 cells were each transfected with a
plasmid encoding green fluorescent protein and either CLCA1 or
gob-5. Individual cells fluorescing green were selected for
electrophysiological measurements to determine if CLCA1 and Gob-5,
in the presence of physiological levels of calcium, could establish
a whole cell chloride current.
[0084] All electrophysiological experiments were performed using an
AxoPatch200B amplifier (Axon Instruments, Foster City, Calif.).
Voltage-clamp protocols and data acquisition were controlled by
pClamp8 software (Axon Instruments). Analog data were filtered at 1
KHz and then digitized at 10 kHz with digitizer DIGIDATA 1321A
(Axon Instruments, Union City, Calif.). All experiments were
performed at room temperature.
[0085] Whole-cell Cl-- currents across the plasma membrane were
isolated pharmacologically by replacing all monovalent cations with
cesium. For conventional whole-cell recording through ruptured
membrane patches, the bath solution contained: 145 mM CsCl, 2 mM
MgCl2, 1 mM CaCl.sub.2, 13 mM glucose, and 10 mM Hepes pH 7.4. The
pipette solution contained: 145 mM CsCl, 1 mM MgCl.sub.2, 1 mM
MgATP, 2 mM BAPTA (or 1 mM Dibromo-BAPTA for 1 .mu.M free
Ca.sup.2+), 10 mM Hepes pH 7.2, and an appropriate concentration of
CaCl.sub.2 (estimated using MaxChelator software, Stanford
University, Palo Alto, Calif.) to give a free [Ca.sup.2+] between
0.1 and 1 .mu.M.
[0086] The whole-cell Cl-- currents were monitored with a protocol
which began by voltage clamping the cell at 0 mV for 30
milliseconds (ms). Then, the voltage was switched to -100 mV for
200 ms, stepped back to 0 mV for 30 ms, then stepped to 100 mV for
200 ms, and finally stepped back to 0 mV. This sequence of voltage
steps was repeated every 5 seconds for 5 minutes to monitor current
development. I/V relationship of Ca.sup.2+ activated whole-cell
Cl-- current was obtained with the following protocol: membrane
potential was held at 0 mV and then 0 stepped to a 300 ms test
pulse ranging from -100 mV to +100 mV (in increments of 20 mV)
before stepping back to 0 mV. All reported current was measured at
5 ms before the end of test pulse and normalized by cell
capacitance. The results presented in FIG. 13a showed that hCLCA1
and gob-5 overexpression in HEK293 and A549 cells does not result
in a whole cell chloride current under physiological levels of
calcium. Moreover, no dose response in current density to
increasing concentrations of calcium was seen (FIG. 13b).
[0087] All references cited herein are incorporated herein by
reference in their entirety and for all purposes to the same extent
as if each individual publication or patent or patent application
was specifically and individually indicated to be incorporated by
reference in its entirety for all purposes. To the extent
publications and patents or patent applications incorporated by
reference contradict the disclosure contained in the specification,
the specification is intended to supercede and/or take precedence
over any such contradictory material.
[0088] All numbers expressing quantities of ingredients, reaction
conditions, and so forth used in the specification and claims are
to be understood as being modified in all instances by the term
"about." Accordingly, unless indicated to the contrary, the
numerical parameters set forth in the specification and attached
claims are approximations that may vary depending upon the desired
properties sought to be obtained by the present invention. At the
very least, and not as an attempt to limit the application of the
doctrine of equivalents to the scope of the claims, each numerical
parameter should be construed in light of the number of significant
digits and ordinary rounding approaches.
[0089] Many modifications and variations of this invention can be
made without departing from its spirit and scope, as will be
apparent to those skilled in the art. The specific embodiments
described herein are offered by way of example only and are not
meant to be limiting in any way. It is intended that the
specification and examples be considered as exemplary only, with
the true scope and spirit of the invention being indicated by the
following claims.
Sequence CWU 1
1
10 1 39 DNA Homo sapiens 1 cgcaagcttg ggatggggcc atttaagagt
tctgtgttc 39 2 39 DNA Homo sapiens 2 cccaagcttt cactacttat
taatttcagg tcttggtgg 39 3 41 DNA Homo sapiens 3 cccaagcttt
cactaccttg agtagacacc gtcatcctta g 41 4 48 DNA Homo sapiens 4
cccaagcttt cactattgat ctgaagcata tgtctgtaaa cctccctg 48 5 15 PRT
Homo sapiens 5 Gly Glu Lys Gly Glu Arg Ile His Leu Thr Pro Asp Phe
Ile Ala 1 5 10 15 6 15 PRT Homo sapiens 6 Glu Gln Asn His Asn Lys
Glu Ala Pro Gln Lys Gln Asn Gln Lys 1 5 10 15 7 17 PRT Homo sapiens
7 Gly Gln Gly Val Ser Gln Arg Ser Ile Gln Leu Glu Ser Lys Gly Leu 1
5 10 15 Thr 8 15 PRT Homo sapiens 8 Ile Glu Asn Asp Glu Ile Gln Trp
Asn Pro Pro Arg Pro Glu Ile 1 5 10 15 9 15 PRT Homo sapiens 9 Lys
Glu Ala Asn Ser Glu Glu Val Phe Leu Phe Lys Pro Glu Asn 1 5 10 15
10 914 PRT Homo sapiens 10 Met Gly Pro Phe Lys Ser Ser Val Phe Ile
Leu Ile Leu His Leu Leu 1 5 10 15 Glu Gly Ala Leu Ser Asn Ser Leu
Ile Gln Leu Asn Asn Asn Gly Tyr 20 25 30 Glu Gly Ile Val Val Ala
Ile Asp Pro Asn Val Pro Glu Asp Glu Thr 35 40 45 Leu Ile Gln Gln
Ile Lys Asp Met Val Thr Gln Ala Ser Leu Tyr Leu 50 55 60 Phe Glu
Ala Thr Gly Lys Arg Phe Tyr Phe Lys Asn Val Ala Ile Leu 65 70 75 80
Ile Pro Glu Thr Trp Lys Thr Lys Ala Asp Tyr Val Arg Pro Lys Leu 85
90 95 Glu Thr Tyr Lys Asn Ala Asp Val Leu Val Ala Glu Ser Thr Pro
Pro 100 105 110 Gly Asn Asp Glu Pro Tyr Thr Glu Gln Met Gly Asn Cys
Gly Glu Lys 115 120 125 Gly Glu Arg Ile His Leu Thr Pro Asp Phe Ile
Ala Gly Lys Lys Leu 130 135 140 Ala Glu Tyr Gly Pro Gln Gly Arg Ala
Phe Val His Glu Trp Ala His 145 150 155 160 Leu Arg Trp Gly Val Phe
Asp Glu Tyr Asn Asn Asp Glu Lys Phe Tyr 165 170 175 Leu Ser Asn Gly
Arg Ile Gln Ala Val Arg Cys Ser Ala Gly Ile Thr 180 185 190 Gly Thr
Asn Val Val Lys Lys Cys Gln Gly Gly Ser Cys Tyr Thr Lys 195 200 205
Arg Cys Thr Phe Asn Lys Val Thr Gly Leu Tyr Glu Lys Gly Cys Glu 210
215 220 Phe Val Leu Gln Ser Arg Gln Thr Glu Lys Ala Ser Ile Met Phe
Ala 225 230 235 240 Gln His Val Asp Ser Ile Val Glu Phe Cys Thr Glu
Gln Asn His Asn 245 250 255 Lys Glu Ala Pro Asn Lys Gln Asn Gln Lys
Cys Asn Leu Arg Ser Thr 260 265 270 Trp Glu Val Ile Arg Asp Ser Glu
Asp Phe Lys Lys Thr Thr Pro Met 275 280 285 Thr Thr Gln Pro Pro Asn
Pro Thr Phe Ser Leu Leu Gln Ile Gly Gln 290 295 300 Arg Ile Val Cys
Leu Val Leu Asp Lys Ser Gly Ser Met Ala Thr Gly 305 310 315 320 Asn
Arg Leu Asn Arg Leu Asn Gln Ala Gly Gln Leu Phe Leu Leu Gln 325 330
335 Thr Val Glu Leu Gly Ser Trp Val Gly Met Val Thr Phe Asp Ser Ala
340 345 350 Ala His Val Gln Ser Glu Leu Ile Gln Ile Asn Ser Gly Ser
Asp Arg 355 360 365 Asp Thr Leu Ala Lys Arg Leu Pro Ala Ala Ala Ser
Gly Gly Thr Ser 370 375 380 Ile Cys Ser Gly Leu Arg Ser Ala Phe Thr
Val Ile Arg Lys Lys Tyr 385 390 395 400 Pro Thr Asp Gly Ser Glu Ile
Val Leu Leu Thr Asp Gly Glu Asp Asn 405 410 415 Thr Ile Ser Gly Cys
Phe Asn Glu Val Lys Gln Ser Gly Ala Ile Ile 420 425 430 His Thr Val
Ala Leu Gly Pro Ser Ala Ala Gln Glu Leu Glu Glu Leu 435 440 445 Ser
Lys Met Thr Gly Gly Leu Gln Thr Tyr Ala Ser Asp Gln Val Gln 450 455
460 Asn Asn Gly Leu Ile Asp Ala Phe Gly Ala Leu Ser Ser Gly Asn Gly
465 470 475 480 Ala Val Ser Gln Arg Ser Ile Gln Leu Glu Ser Lys Gly
Leu Thr Leu 485 490 495 Gln Asn Ser Gln Trp Met Asn Gly Thr Val Ile
Val Asp Ser Thr Val 500 505 510 Gly Lys Asp Thr Leu Phe Leu Ile Thr
Trp Thr Thr Gln Pro Pro Gln 515 520 525 Ile Leu Leu Trp Asp Pro Ser
Gly Gln Lys Gln Gly Gly Phe Val Val 530 535 540 Asp Lys Asn Thr Lys
Met Ala Tyr Leu Gln Ile Pro Gly Ile Ala Lys 545 550 555 560 Val Gly
Thr Trp Lys Tyr Ser Leu Gln Ala Ser Ser Gln Thr Leu Thr 565 570 575
Leu Thr Val Thr Ser Arg Ala Ser Asn Ala Thr Leu Pro Pro Ile Thr 580
585 590 Val Thr Ser Lys Thr Asn Lys Asp Thr Ser Lys Phe Pro Ser Pro
Leu 595 600 605 Val Val Tyr Ala Asn Ile Arg Gln Gly Ala Ser Pro Ile
Leu Arg Ala 610 615 620 Ser Val Thr Ala Leu Ile Glu Ser Val Asn Gly
Lys Thr Val Thr Leu 625 630 635 640 Glu Leu Leu Asp Asn Gly Ala Gly
Ala Asp Ala Thr Lys Asp Asp Gly 645 650 655 Val Tyr Ser Arg Tyr Phe
Thr Thr Tyr Asp Thr Asn Gly Arg Tyr Ser 660 665 670 Val Lys Val Arg
Ala Leu Gly Gly Val Asn Ala Ala Arg Arg Arg Val 675 680 685 Ile Pro
Gln Gln Ser Gly Ala Leu Tyr Ile Pro Gly Trp Ile Glu Asn 690 695 700
Asp Glu Ile Gln Trp Asn Pro Pro Arg Pro Glu Ile Asn Lys Asp Asp 705
710 715 720 Val Gln His Lys Gln Val Cys Phe Ser Arg Thr Ser Ser Gly
Gly Ser 725 730 735 Phe Val Ala Ser Asp Val Pro Asn Ala Pro Ile Pro
Asp Leu Phe Pro 740 745 750 Pro Gly Gln Ile Thr Asp Leu Lys Ala Glu
Ile His Gly Gly Ser Leu 755 760 765 Ile Asn Leu Thr Trp Thr Ala Pro
Gly Asp Asp Tyr Asp His Gly Thr 770 775 780 Ala His Lys Tyr Ile Ile
Arg Ile Ser Thr Ser Ile Leu Asp Leu Arg 785 790 795 800 Asp Lys Phe
Asn Glu Ser Leu Gln Val Asn Thr Thr Ala Leu Ile Pro 805 810 815 Lys
Glu Ala Asn Ser Glu Glu Val Phe Leu Phe Lys Pro Glu Asn Ile 820 825
830 Thr Phe Glu Asn Gly Thr Asp Leu Phe Ile Ala Ile Gln Ala Val Asp
835 840 845 Lys Val Asp Leu Lys Ser Glu Ile Ser Asn Ile Ala Arg Val
Ser Leu 850 855 860 Phe Ile Pro Pro Gln Thr Pro Pro Glu Thr Pro Ser
Pro Asp Glu Thr 865 870 875 880 Ser Ala Pro Cys Pro Asn Ile His Ile
Asn Ser Thr Ile Pro Gly Ile 885 890 895 His Ile Leu Lys Ile Met Trp
Lys Trp Ile Gly Glu Leu Gln Leu Ser 900 905 910 Ile Ala
* * * * *
References